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Publication numberUS2277716 A
Publication typeGrant
Publication dateMar 31, 1942
Filing dateJul 8, 1940
Priority dateJul 8, 1940
Publication numberUS 2277716 A, US 2277716A, US-A-2277716, US2277716 A, US2277716A
InventorsLewis B Lockwood, George E Ward, Joseph J Stubbs, Edward T Roe, Tabenkin Benjamin
Original AssigneeHenry A Wallace
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Fermentation process for the production of 2-ketogluconic acid
US 2277716 A
Abstract  available in
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Description  (OCR text may contain errors)

Patented Mar. 31 1942 'FERMENTATION rnoonss Fon'r m PRO- DUCTION or z-xn'roowcomc ACID Lewis B. Lockwood, Alexandria, and George E. Ward, Arlington, Va., and Joseph J. Stubbs, Washington, D. C., and Edward T. Roe, Arlington, and Benjamin Tabenkin, Alexandria, Va., assignors to Henry A. Wallace, as Secretary of Agriculture 01' the United States of America, and to his successors in ofli'ce No Drawing. Applicaiign July 8, 1940, Serial No. a 3 o i 7 Claims. (01. 195-47) (Granted under the act of March 3, 1883, as amended April 30, 1928; 370 0. 757) This application is' made under the act of March 3, 1883, as amended by the act of April 30, 1928, and the invention herein described and claimed, if patented, may be manufactured-and used by or for the Government of the United States of America for governmental purposes without the payment to us of any royalty thereon;

This invention relates to a method for the preparation of ketocarboxylic acids, and more particularly to a method for the preparation, by

fermentation, of a valuab1e.material which finds use as an intermediate for the preparation of d- -isoascorbic acid (also known as d-araboascorbic acid), which possesses marked antiscorbutic and antioxidant properties. (Maurer and Schiedt, Ber. der deutsch. chem. Gesell. vol. 66, page 1054 (1933.) and vol. 67, page 1239 (1934); also Ohle, U. S. Patent 2,160,621 (1939); also Dalmer and Moll, Zeitschr. 'physiol, Chemie, vol. 222, page The production of 2*ketog1uconi'c acid by fermentation methods has been only recently observed. Thus, in 1935, Bernhauer and Giirlich (Biochemlsche Zeitschrift 280, 357) isolated small quantities of 2-ketogluconic acid'from cultures of Acetobacter gluconicum grown on solutions of calcium gluconate, and in 1938, Bernhauer and -Knobloch (Naturwissenschaften 50,819) obtained 2-ketogluconic acid from cultures of Acetobacter suboxydans grown on aqueous solutions of salts fermentation periods of 20 days or more, it is possible, by applying our invention, to obtain 80 to 95% yields of 2-ketogluconic acid in 30 to 40 hours. vA further advantage of our invention is that we are not restricted to the use of gluconate salts as substrates, as heretofore, but we may use glucose or carbohydrate materials containing glucose as a. component of the sugar molecule, such as maltose, dextrins, starch, molasses, grain mashes, and the like. A still further advantage of our invention is to be found in the fact that no' -ketogluconic acid is formed, as has been heretofore encountered.

We have found that numerous bacteria of the genus Pseudornonas possess the ability to produce 2-ketogluconic acid from. the above-mentioned substrates, when cultivated according tothe methods herein set forth. Thus, we have found "that many strains distributed among the followof gluconic acid. In both of these cases the bacteria were grown in thin films or pellicles in unagitated surface cultures and the conversion of substrate to product required twenty days, or more. The reaction was complicated by the simultaneous formation of 5-ket0g1uconic acid.

The formation of 2-ketogluconic acid was-also erably at superatmospheric pressure, thesystem at the same. time being agitated by various means, a rapid and eiiicient. conversion of substrate to Z-ketogluconic acid occurs. In contrast to prel at super-atmospheric pressure.

ing species may be used to effect this conversion: Pseudomonas schuylkilliensis, Ps. putida, Ps. graveolens, Ps. vendz'elli, Ps. ovalis, Pajragii, Ps. mildenbergii, Ps. mucidolens, and Ps. fluorescens, It is thus apparent that the ability to produce 2-ketogluconic acid is characteristic of the genus Pseudomonas taken as a whole, when the bacteria are cultivated in the manner taught by us.-

' In our invention, agitation of the system may be effected by blowing air through the mass, or by propellers, or by revolving the fermenter, or by other means which will occur to those skilled in the art. The exact apparatus used to effect agitation is not critical, the important factor being the intimate contacting of the bacterial cells, ,the substrate, the neutralizing agent, and the gas used for aeration.

We have found that aeration of the mash is necessary to obtain a rapid oxidation of the substrate to 2-ket'ogluconic acid. Such aeration may be applied at atmospheric pressure, or preferably, We have found that operating at super-atmospheric pressure re-' sults in a more rapid conversion of substrate to product. I

We have found it desirable to conduct our fermentation at temperatures between 20 C. and C., the range from 25 C. to 30 C. being especially suitable.

vious mediocre yields of 2-ketogluconic 3 acid in 50 As typical apparatus within which our process vention as to equipment used, since numerous modifications and adaptations are possible, and will be readily apparent to those skilled in the art.

Considerable latitude is possible in the selection of nutrients to be used in performing our invention. Although we prefer touse corn steeping liquor, urea, magnesium sulfate, and potassium phosphate, as cited hereatfer in Example 1, it is possible to secure good results if some of these components are omitted or varied as to the quantity used. Thus, an entirely satisfac- .tory fermentation may be obtained upon omitting the urea, magnesium sulfate and potassium phosphate, if the quantity of corn steeping liquor be increased. Similarly, if urea and the abovementioned inorganic salts be supplied in the quantities given in Example 1, the corn steeping liquor may be reduced in quanity or even entirely eliminated without noticeably affecting the fermentation. We accordingly do not wish to be restricted as to the nutrient components, since many variations will be apparent to those skilled in the art. Likewise, we do not wish to'be restricted as to the use of calcium carbonate as the neutralizing agent, since the substitution of quicklime, zinc carbonate, and other similar substances will readily occur to those skilled in the art. 1

The following examples illustrate representative procedures used in practicing our invention:'

Example 1 An aqueous fermentation medium of the following compositionwas used:

' Grams per liter of medium Glucose 100. Corn steeping liquor- 5. Octadecyl alcohol 0.3 Urea 2.0 MgSO4.7H2O -l 0.25 KHzPOi 0.60 CaCO: 27.0

3200 cc. of this sterile medium were inoculated with approximately 300 cc. of an active culture of Pseudomonas flum'escens and placed in a 1'0- tary drum fermenter. Air at atmosphericfiiressure was passed through through the fermenter at a rate of 1600 cc. per minute, the solution was agitated by revolving the drum l3 revolutions per minute, and the temperature of the system was maintained at approximately 25 C. The course of the fermentation was followed by periodic analysis. After 43 hours, the glucose was all consumed and 2-ketogluconic acid (calcium salt) was present in a quantity equivalent to a 72% yield, based on the glucose available. The prod-' not was identified by its optical properties and by preparation of the methyl ester (M. P. 174 C.).-

Elramplc 2 The same materials and conditions were used as in Example 1, except that the process was conducted under increased air pressure, a gage pressure of 30 pounds per square 'inch being maintained. After 25 hours, the glucose was all consumed and 2-ketogluconic acid (calcium salt) was present in a quantity equivalent to an 81% yield based on the glucose available.

Example 3 The same materials and conditions were used as in Example 2, except that calcium gluconate (50 grains per liter) was used as the substrate instead of glucose. After hours, a 63% yield of 2-ketogluconic acid (calcium salt) was obtained.

Example 4 The same materials and conditions were used as in Example 2, except that potassium gluconate (50 grams per liter) was used as the substrate. After 65 hours a 61.5% yield of 2-ketogluconic acid was obtained.

Example 5 A fermentation medium of the composition given in Example 1 was distributed in 200 cc. portions of Jena glass gas-washing bottles (type 1010). Following sterilization, individual bottles were inoculated with selected species of Pseudomonas. Each bottle was then aerated for 8 days with sterile, humidified air at a rate of approximately 200 cc. per minute, the incubation temperature being 30 C. throughout. The species used, and the yields of 2-ketogluconic acid (calcium salt) obtained were as follows:

-, ,32 liters of a fermentation medium of the composition given in Example 1 was sterilized inavertical aluminum vat equipped with agitating means, and means for dispersing air. The octadecyl alcoho], supplemented by small portions of lard oil, eliminated frothing and foaming during the fermentation. The nutrient solution was inocu-' lated with an active culture of Pseudomonas fluorescens and maintained at 25 C., with agitation as'above described, and aeration at 12 to 16 iiters of air per minute. The system was maintained at 30 pounds gage pressure. After 43 hours the glucose was'all consumed and an 88% Having thus described our invention, what we claim for Letters Patent is:

1. A process for the production of 2-ketoglu conic acid, which comprises inoculating a carbohydrate mash with bacteria of the genus Pseudomonas; thence aerating and agitating the inoculated mash, the while cultivating the bacteria in a submerged state, thereby producing Z-ketogluconic aci'd.

2. A process for the production of 2-ketoglu Yield conic acid, which comprises inoculating a carbohydrate mash with bacteria of the genus Pseudomonas, thence aerating and agitating the inoculated mash, .the while maintaining the same under super-atmospheric pressure of gases containing substantial quantities. of oxygen, and cul Pseudomonas, thence aerating and agitating the inoculated mash, the while maintaining the same under superatmospheric pressure of gases containing substantial quantities of oxygen, and cultivating the bacteria in a submerged state, thereby producing 2-ketogluconic acid.

5. A process for the production of 2-ketogluconic acid, which comprises inoculating a nutri-* ent solution containing a gluconate salt with bacteria of the genus Pseudomonas; thence aerating and agitating the inoculated mash, the while cultivating the bacteria in a submerged state,

thereby producing Z-ketogluconic acid.

6. A process for the production of 2-ketogluconic acid, which comprises inoculating a nutrient solution containing a gluconate salt with bacteria of the genus Pseudomonas, thence aerating and agitating the inocu1ated mash, the while maintaining the same under super-atmospheric pressure of-gases containing. substantial quantitles of oxygen, and cultivating the bacteria in a "submerged state, thereby producing 2-ketogluconic acid.

f'l. A process for the production of 2-ketogluconic acid, which comprises inoculating a carbohydrate mash with bacteria of the group consisting of the species Pseudomonas fluorescens, Ps. fragii, Ps. graveolens, Ps. mildenbergii, Ps.

' mucidolens, Ps. putida, Ps. schuylkilliensis, Ps.

vendrelli, and Ps. ovalis; thence aerating and agitating the inoculated mash, the while cultivating the bacteria in a submerged state, thereby producing2-ketogluconic acid,

' LEWIS B. LOCKWOOD.

GEORGE E. WARD. JOSEPH J. STUBBS. EDWARD T. ROE; BENJAIVHN TABENKIN.

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US2422230 *Sep 11, 1943Jun 17, 1947Merck & Co IncProduction of streptothricin
US2496297 *Apr 11, 1947Feb 7, 1950Lockwood Lewis ByfordProcess of culturing bacteria
US2550450 *Jun 6, 1947Apr 24, 1951Swift & CoDefoamer
US2559650 *Oct 28, 1949Jul 10, 1951Lewis B LockwoodPreparation of d-tartaric acid
US2594283 *Dec 29, 1949Apr 29, 1952Us AgricultureProcess for the preparation of inoculum for use in the fermentative production of sodium gluconate
US2707683 *Dec 11, 1951May 3, 1955Heinz EbnerMethod for the production of vinegar acids by oxidative fermentation of alcohols
US2724680 *Mar 11, 1952Nov 22, 1955Harold J KoepsellPreparation and recovery of alpha-ketoglutaric acid
US2917435 *Sep 27, 1956Dec 15, 1959Olin MathiesonPreparation of 2-keto-1-gulonic acid by pseudomonas aeruginosa
US2918492 *Aug 24, 1956Dec 22, 1959Miles LabMethod of preparing methyl 2-ketogluconate
US3255093 *Mar 5, 1964Jun 7, 1966Merck & Co IncConversion of glucose to 2-ketogluconic acid
US4246348 *Oct 23, 1978Jan 20, 1981Novo Laboratories, Inc.Microbiologic conversion of L-galactonate into 2-Keto L-galactonate
US4892823 *Jun 4, 1987Jan 9, 1990Takeda Chemical Industries, Ltd.Method for producing 2-keto-L-gulonic acid
US4933289 *Sep 27, 1989Jun 12, 1990Takeda Chemical Industries, Ltd.Biologically pure cultures of Pseudomonas sorbosoxidans useful for producing 2-keto-L-gulonic acid
EP0007751A1 *Jul 13, 1979Feb 6, 1980Pfizer Inc.Destruction by fermentation of 2-ketogluconate in the presence of 2-ketogulonate
Classifications
U.S. Classification435/138, 435/877, 435/876
International ClassificationC07H7/027, C12P7/58
Cooperative ClassificationC12P7/58, Y10S435/877, C07H7/027, Y10S435/876
European ClassificationC12P7/58, C07H7/027